Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Jul;65(7):2648–2655. doi: 10.1128/iai.65.7.2648-2655.1997

Nitrite reductase from Pseudomonas aeruginosa induces inflammatory cytokines in cultured respiratory cells.

K Oishi 1, B Sar 1, A Wada 1, Y Hidaka 1, S Matsumoto 1, H Amano 1, F Sonoda 1, S Kobayashi 1, T Hirayama 1, T Nagatake 1, K Matsushima 1
PMCID: PMC175374  PMID: 9199432

Abstract

Persistent infection with Pseudomonas aeruginosa increases interleukin-8 (IL-8) levels and causes dense neutrophil infiltrations in the airway of patients with chronic airway diseases. To investigate the role of P. aeruginosa infection in IL-8 production in the airway of these patients, we examined whether cell lysates of P. aeruginosa could cause IL-8 production from human bronchial epithelial cells. Diluted sonicated supernatants of P. aeruginosa (SSPA) with a mucoid or nonmucoid phenotype stimulated human bronchial epithelial (BET-1A) cells to produce IL-8. In this study, we have purified a 59-kDa heat-stable protein with IL-8-inducing activity from the SSPA by sequential ion-exchange chromatography. The N-terminal sequence of this purified protein completely matched a sequence at the N-terminal part of the mature protein of nitrite reductase from P. aeruginosa. In addition, immunoblotting with a polyclonal immunoglobulin G (IgG) against recombinant Pseudomonas nitrite reductase demonstrated a specific binding to the purified protein. Furthermore, the immunoprecipitates of the SSPA with a polyclonal IgG against recombinant nitrite reductase induced a twofold-higher IL-8 production in the BET-1A cell culture than did the immunoprecipitates of the SSPA with a control IgG. These lines of evidence confirmed that Pseudomonas nitrite reductase was responsible for IL-8 production in the BET-1A cells. The purified nitrite reductase induced maximal expression of IL-8 mRNA in the BET-1A cells at 1 to 3 h after stimulation, and the IL-8 mRNA expression declined by 8 h after stimulation. New protein translation was not required for nitrite reductase-mediated IL-8 mRNA expression in the BET-1A cells. Nitrite reductase stimulated the BET-1A cells, as well as human alveolar macrophages, pulmonary fibroblasts, and neutrophils, to produce IL-8. In contrast, nitrite reductase induced significant levels of tumor necrosis factor alpha and IL-1beta protein only in human alveolar macrophages. These data support the notion that nitrite reductase from P. aeruginosa induces the production of inflammatory cytokines by respiratory cells and may contribute to the pathogenesis of chronic airway diseases and persistent P. aeruginosa infection.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrew P. J., Harant H., Lindley I. J. Nitric oxide regulates IL-8 expression in melanoma cells at the transcriptional level. Biochem Biophys Res Commun. 1995 Sep 25;214(3):949–956. doi: 10.1006/bbrc.1995.2378. [DOI] [PubMed] [Google Scholar]
  2. Chalkley L. J., Koornhof H. J. Antimicrobial activity of ciprofloxacin against Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus determined by the killing curve method: antibiotic comparisons and synergistic interactions. Antimicrob Agents Chemother. 1985 Aug;28(2):331–342. doi: 10.1128/aac.28.2.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Coyne M. S., Arunakumari A., Averill B. A., Tiedje J. M. Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria. Appl Environ Microbiol. 1989 Nov;55(11):2924–2931. doi: 10.1128/aem.55.11.2924-2931.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Coyne M. S., Arunakumari A., Pankratz H. S., Tiedje J. M. Localization of the cytochrome cd1 and copper nitrite reductases in denitrifying bacteria. J Bacteriol. 1990 May;172(5):2558–2562. doi: 10.1128/jb.172.5.2558-2562.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DiMango E., Zar H. J., Bryan R., Prince A. Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J Clin Invest. 1995 Nov;96(5):2204–2210. doi: 10.1172/JCI118275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eigler A., Sinha B., Endres S. Nitric oxide-releasing agents enhance cytokine-induced tumor necrosis factor synthesis in human mononuclear cells. Biochem Biophys Res Commun. 1993 Oct 15;196(1):494–501. doi: 10.1006/bbrc.1993.2277. [DOI] [PubMed] [Google Scholar]
  7. Fick R. B., Jr Pathogenesis of the pseudomonas lung lesion in cystic fibrosis. Chest. 1989 Jul;96(1):158–164. doi: 10.1378/chest.96.1.158. [DOI] [PubMed] [Google Scholar]
  8. Francoeur C., Denis M. Nitric oxide and interleukin-8 as inflammatory components of cystic fibrosis. Inflammation. 1995 Oct;19(5):587–598. doi: 10.1007/BF01539138. [DOI] [PubMed] [Google Scholar]
  9. Fülöp V., Moir J. W., Ferguson S. J., Hajdu J. The anatomy of a bifunctional enzyme: structural basis for reduction of oxygen to water and synthesis of nitric oxide by cytochrome cd1. Cell. 1995 May 5;81(3):369–377. doi: 10.1016/0092-8674(95)90390-9. [DOI] [PubMed] [Google Scholar]
  10. HORIO T., HIGASHI T., YAMANAKA T., MATSUBARA H., OKUNUKI K. Purification and properties of cytochrome oxidase from Pseudomonas aeruginosa. J Biol Chem. 1961 Mar;236:944–951. [PubMed] [Google Scholar]
  11. Hoiby N. Pseudomonas aeruginosa infection in cystic fibrosis. Diagnostic and prognostic significance of pseudomonas aeruginosa precipitins determined by means of crossed immunoelectrophoresis. A survey. Acta Pathol Microbiol Scand Suppl. 1977;(262):1–96. [PubMed] [Google Scholar]
  12. Homma H., Yamanaka A., Tanimoto S., Tamura M., Chijimatsu Y., Kira S., Izumi T. Diffuse panbronchiolitis. A disease of the transitional zone of the lung. Chest. 1983 Jan;83(1):63–69. doi: 10.1378/chest.83.1.63. [DOI] [PubMed] [Google Scholar]
  13. Howarth P. H., Bradding P., Montefort S., Peroni D., Djukanovic R., Carroll M. P., Holgate S. T. Mucosal inflammation and asthma. Am J Respir Crit Care Med. 1994 Nov;150(5 Pt 2):S18–S22. doi: 10.1164/ajrccm/150.5_Pt_2.S18. [DOI] [PubMed] [Google Scholar]
  14. Inoue H., Hara M., Massion P. P., Grattan K. M., Lausier J. A., Chan B., Kaneko T., Isono K., Jorens P. G., Ueki I. F. Role of recruited neutrophils in interleukin-8 production in dog trachea after stimulation with Pseudomonas in vivo. Am J Respir Cell Mol Biol. 1995 Nov;13(5):570–577. doi: 10.1165/ajrcmb.13.5.7576693. [DOI] [PubMed] [Google Scholar]
  15. Inoue H., Massion P. P., Ueki I. F., Grattan K. M., Hara M., Dohrman A. F., Chan B., Lausier J. A., Golden J. A., Nadel J. A. Pseudomonas stimulates interleukin-8 mRNA expression selectively in airway epithelium, in gland ducts, and in recruited neutrophils. Am J Respir Cell Mol Biol. 1994 Dec;11(6):651–663. doi: 10.1165/ajrcmb.11.6.7946394. [DOI] [PubMed] [Google Scholar]
  16. Joiner K. A. Studies on the mechanism of bacterial resistance to complement-mediated killing and on the mechanism of action of bactericidal antibody. Curr Top Microbiol Immunol. 1985;121:99–133. doi: 10.1007/978-3-642-45604-6_6. [DOI] [PubMed] [Google Scholar]
  17. Kadota J., Sakito O., Kohno S., Sawa H., Mukae H., Oda H., Kawakami K., Fukushima K., Hiratani K., Hara K. A mechanism of erythromycin treatment in patients with diffuse panbronchiolitis. Am Rev Respir Dis. 1993 Jan;147(1):153–159. doi: 10.1164/ajrccm/147.1.153. [DOI] [PubMed] [Google Scholar]
  18. Ko Y., Mukaida N., Panyutich A., Voitenok N. N., Matsushima K., Kawai T., Kasahara T. A sensitive enzyme-linked immunosorbent assay for human interleukin-8. J Immunol Methods. 1992 May 18;149(2):227–235. doi: 10.1016/0022-1759(92)90254-q. [DOI] [PubMed] [Google Scholar]
  19. König B., Ceska M., König W. Effect of Pseudomonas aeruginosa on interleukin-8 release from human phagocytes. Int Arch Allergy Immunol. 1995 Apr;106(4):357–365. doi: 10.1159/000236867. [DOI] [PubMed] [Google Scholar]
  20. Lesage D., Delisle F., Richard G., Burghoffer B., Le Cunff D., Petit J. C. Comparison of two techniques for measurement of in vitro killing kinetics of five antibiotics against Pseudomonas aeruginosa. Eur J Clin Microbiol Infect Dis. 1994 May;13(5):412–417. doi: 10.1007/BF01972000. [DOI] [PubMed] [Google Scholar]
  21. Massion P. P., Inoue H., Richman-Eisenstat J., Grunberger D., Jorens P. G., Housset B., Pittet J. F., Wiener-Kronish J. P., Nadel J. A. Novel Pseudomonas product stimulates interleukin-8 production in airway epithelial cells in vitro. J Clin Invest. 1994 Jan;93(1):26–32. doi: 10.1172/JCI116954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Matsushima K., Morishita K., Yoshimura T., Lavu S., Kobayashi Y., Lew W., Appella E., Kung H. F., Leonard E. J., Oppenheim J. J. Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J Exp Med. 1988 Jun 1;167(6):1883–1893. doi: 10.1084/jem.167.6.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McElvaney N. G., Nakamura H., Birrer P., Hébert C. A., Wong W. L., Alphonso M., Baker J. B., Catalano M. A., Crystal R. G. Modulation of airway inflammation in cystic fibrosis. In vivo suppression of interleukin-8 levels on the respiratory epithelial surface by aerosolization of recombinant secretory leukoprotease inhibitor. J Clin Invest. 1992 Oct;90(4):1296–1301. doi: 10.1172/JCI115994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nakamura H., Yoshimura K., Jaffe H. A., Crystal R. G. Interleukin-8 gene expression in human bronchial epithelial cells. J Biol Chem. 1991 Oct 15;266(29):19611–19617. [PubMed] [Google Scholar]
  25. Nakamura H., Yoshimura K., McElvaney N. G., Crystal R. G. Neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-8 gene expression in a human bronchial epithelial cell line. J Clin Invest. 1992 May;89(5):1478–1484. doi: 10.1172/JCI115738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Obayashi T., Tamura H., Tanaka S., Ohki M., Takahashi S., Kawai T. Endotoxin-inactivating activity in normal and pathological human blood samples. Infect Immun. 1986 Aug;53(2):294–297. doi: 10.1128/iai.53.2.294-297.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Oishi K., Sonoda F., Kobayashi S., Iwagaki A., Nagatake T., Matsushima K., Matsumoto K. Role of interleukin-8 (IL-8) and an inhibitory effect of erythromycin on IL-8 release in the airways of patients with chronic airway diseases. Infect Immun. 1994 Oct;62(10):4145–4152. doi: 10.1128/iai.62.10.4145-4152.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Persoons J. H., Schornagel K., Tilders F. F., De Vente J., Berkenbosch F., Kraal G. Alveolar macrophages autoregulate IL-1 and IL-6 production by endogenous nitric oxide. Am J Respir Cell Mol Biol. 1996 Mar;14(3):272–278. doi: 10.1165/ajrcmb.14.3.8845178. [DOI] [PubMed] [Google Scholar]
  29. Pier G. B., Ames P. Mediation of the killing of rough, mucoid isolates of Pseudomonas aeruginosa from patients with cystic fibrosis by the alternative pathway of complement. J Infect Dis. 1984 Aug;150(2):223–228. doi: 10.1093/infdis/150.2.223. [DOI] [PubMed] [Google Scholar]
  30. Ponglertnapagorn P., Oishi K., Iwagaki A., Sonoda F., Watanabe K., Nagatake T., Matsushima K., Matsumoto K. Airway interleukin-8 in elderly patients with bacterial lower respiratory tract infections. Microbiol Immunol. 1996;40(2):177–182. doi: 10.1111/j.1348-0421.1996.tb03322.x. [DOI] [PubMed] [Google Scholar]
  31. Richman-Eisenstat J. B., Jorens P. G., Hébert C. A., Ueki I., Nadel J. A. Interleukin-8: an important chemoattractant in sputum of patients with chronic inflammatory airway diseases. Am J Physiol. 1993 Apr;264(4 Pt 1):L413–L418. doi: 10.1152/ajplung.1993.264.4.L413. [DOI] [PubMed] [Google Scholar]
  32. Rolfe M. W., Kunkel S. L., Standiford T. J., Chensue S. W., Allen R. M., Evanoff H. L., Phan S. H., Strieter R. M. Pulmonary fibroblast expression of interleukin-8: a model for alveolar macrophage-derived cytokine networking. Am J Respir Cell Mol Biol. 1991 Nov;5(5):493–501. doi: 10.1165/ajrcmb/5.5.493. [DOI] [PubMed] [Google Scholar]
  33. Sharma S. A., Tummuru M. K., Miller G. G., Blaser M. J. Interleukin-8 response of gastric epithelial cell lines to Helicobacter pylori stimulation in vitro. Infect Immun. 1995 May;63(5):1681–1687. doi: 10.1128/iai.63.5.1681-1687.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Silvestrini M. C., Galeotti C. L., Gervais M., Schininà E., Barra D., Bossa F., Brunori M. Nitrite reductase from Pseudomonas aeruginosa: sequence of the gene and the protein. FEBS Lett. 1989 Aug 28;254(1-2):33–38. doi: 10.1016/0014-5793(89)81004-x. [DOI] [PubMed] [Google Scholar]
  35. Standiford T. J., Kunkel S. L., Kasahara K., Milia M. J., Rolfe M. W., Strieter R. M. Interleukin-8 gene expression from human alveolar macrophages: the role of adherence. Am J Respir Cell Mol Biol. 1991 Dec;5(6):579–585. doi: 10.1165/ajrcmb/5.6.579. [DOI] [PubMed] [Google Scholar]
  36. Villarete L. H., Remick D. G. Nitric oxide regulation of IL-8 expression in human endothelial cells. Biochem Biophys Res Commun. 1995 Jun 15;211(2):671–676. doi: 10.1006/bbrc.1995.1864. [DOI] [PubMed] [Google Scholar]
  37. YAMANAKA T., KIJIMOTO S., OKUNUKI K. Biological significance of Pseudomonas cytochrome oxidase in Pseudomonas aeruginosa. J Biochem. 1963 May;53:416–421. doi: 10.1093/oxfordjournals.jbchem.a127716. [DOI] [PubMed] [Google Scholar]
  38. YAMANAKA T., OKUNUKI K. Crystalline Pseudomonas cytochrome oxidase. I. Enzymic properties with special reference to the biological specificity. Biochim Biophys Acta. 1963 Mar 12;67:379–393. doi: 10.1016/0006-3002(63)91844-4. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES